Formation of Lymphoma Hybrid Spheroids and Drug Testing in Real Time with the Use of Fluorescence Optical Tweezers.

Interactions between stromal and lymphoma cells in the bone marrow are closely related to drug resistance and therapy failure. Physiologically relevant pre-clinical three-dimensional (3D) models recapitulating lymphoma microenvironmental complexity do not currently exist. In this study, we proposed a scheme for optically controlled hybrid lymphoma spheroid formation with the use of optical tweezers (OT). Following the preparation of stromal spheroids using agarose hydrogel, two aggressive non-Hodgkin lymphoma B-cell lines, Ri-1 (DLBCL) and Raji (Burkitt lymphoma), were used to conduct multi-cellular spheroid formation driven by in-house-developed fluorescence optical tweezers. Importantly, the newly formed hybrid spheroid preserved the 3D architecture for the next 24 h. Our model was successfully used for the evaluation of the influence of the anticancer agents doxorubicin (DOX), ibrutinib (IBR), and AMD3100 (plerixafor) on the adhesive properties of lymphoma cells. Importantly, our study revealed that a co-treatment of DOX and IBR with AMD3100 affects the adhesion of B-NHL lymphoma cells.

Optical vortex torque measured with optically trapped microbarbells.

Optical vortex beams carry orbital angular momentum and thus exert torque on illuminated objects. A dielectric microtool-a microbarbell-is used in two-laser optical tweezers to measure the torque of a focused optical vortex. The tool was either freely rotating due to the applied torque or set into oscillations by the counteracting force. Four different trapping configurations provided different ways of sensing the torque and gave consistent results. The value of torque was determined by confronting the experimental results with numerical and analytical models.

Profound Nanoscale Structural and Biomechanical Changes in DNA Helix upon Treatment with Anthracycline Drugs.

In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.

Polarimeter for measuring the properties of birefringent media in reflective mode.

This work presents a description of a polarimetric system for measuring the properties of birefringent media. In our reflection system the applied Stokes polarimeter acts both as a generator of the light's selected polarization states as well as a light analyzer leaving the examined medium. The method is based on six intensity distribution measurements realized in six different configurations of polarizers/analyzers: four linear and two circular ones. Thus, we have achieved parallel polariscope for linear polarizers and the crossed polariscope for circular polarizers. Such a setup can be easily applied for linearly birefringent media properties measurements including dichroic ones. This measurement setup and the measurement method were successfully tested in a homogeneous medium and a medium with variable phase difference.

Differentiation of single lymphoma primary cells and normal B-cells based on their adhesion to mesenchymal stromal cells in optical tweezers.

We have adapted a non-invasive method based on optical tweezers technology to differentiate between the normal B-cells and the B-cell non-Hodgkin lymphoma (B-NHL) cells derived from clinical samples. Our approach bases on the nascent adhesion between an individual B-cell and a mesenchymal stromal cell. In this study, a single B-cell was trapped and optically seeded on a mesenchymal stromal cell and kept in a direct contact with it until a stable connection between the cells was formed in time scale. This approach allowed us to avoid the introduction of any exogenous beads or chemicals into the experimental setup which would have affected the cell-to-cell adhesion. Here, we have provided new evidence that aberrant adhesive properties found in transformed B-cells are related to malignant neoplasia. We have demonstrated that the mean time required for establishing adhesive interactions between an individual normal B-cell and a mesenchymal stromal cell was 26.7 ± 16.6 s, while for lymphoma cell it was 208.8 ± 102.3 s, p < 0.001. The contact time for adhesion to occur ranged from 5 to 90 s and from 60 to 480 s for normal B-cells and lymphoma cells, respectively. This method for optically controlled cell-to-cell adhesion in time scale is beneficial to the successful differentiation of pathological cells from normal B-cells within the fine needle aspiration biopsy of a clinical sample. Additionally, variations in time-dependent adhesion among subtypes of B-NHL, established here by the optical trapping, confirm earlier results pertaining to cell heterogeneity.

Micro-Dumbbells-A Versatile Tool for Optical Tweezers.

Manipulation of micro- and nano-sized objects with optical tweezers is a well-established, albeit still evolving technique. While many objects can be trapped directly with focused laser beam(s), for some applications indirect manipulation with tweezers-operated tools is preferred. We introduce a simple, versatile micro-tool operated with holographic optical tweezers. The 40 µm long dumbbell-shaped tool, fabricated with two-photon laser 3D photolithography has two beads for efficient optical trapping and a probing spike on one end. We demonstrate fluids viscosity measurements and vibration detection as examples of possible applications.

Physiological Hypoxia (Physioxia) Impairs the Early Adhesion of Single Lymphoma Cell to Marrow Stromal Cell and Extracellular Matrix. Optical Tweezers Study.

Adhesion is critical for the maintenance of cellular structures as well as intercellular communication, and its dysfunction occurs prevalently during cancer progression. Recently, a growing number of studies indicated the ability of oxygen to regulate adhesion molecules expression, however, the influence of physiological hypoxia (physioxia) on cell adhesion remains elusive. Thus, here we aimed: (i) to develop an optical tweezers based assay to precisely evaluate single diffuse large B-cell lymphoma (DLBCL) cell adhesion to neighbor cells (mesenchymal stromal cells) and extracellular matrix (Matrigel) under normoxia and physioxia; and, (ii) to explore the role of integrins in adhesion of single lymphoma cell. We identified the pronouncedly reduced adhesive properties of lymphoma cell lines and primary lymphocytes B under physioxia to both stromal cells and Matrigel. Corresponding effects were shown in bulk adhesion assays. Then we emphasized that impaired ß1, ß2 integrins, and cadherin-2 expression, studied by confocal microscopy, account for reduction in lymphocyte adhesion in physioxia. Additionally, the blockade studies conducted with anti-integrin antibodies have revealed the critical role of integrins in lymphoma adhesion. To summarize, the presented approach allows for precise confirmation of the changes in single cell adhesion properties provoked by physiological hypoxia. Thus, our findings reveal an unprecedented role of using physiologically relevant oxygen conditioning and single cell adhesion approaches when investigating tumor adhesion in vitro.

Alterations of biomechanics in cancer and normal cells induced by doxorubicin.

Mechanical properties of biological structures play an important role in regulating cellular activities and are critical for understanding metabolic processes in cancerous cells and the effects of drugs. For some cancers, such as acute myeloid leukaemia, chemotherapy is one of preferential methods. However, due to the lack of selectivity to cancer cells, cytostatic agents cause toxicity to normal tissues. Here, we study the effect of doxorubicin (DOX) on the mechanical properties of DNA molecules, leukemic blast cells and erythrocytes, using optical tweezers. In addition, we controlled the subcellular distribution of the drug by confocal microscopy. Our results indicated that doxorubicin affects mechanical properties of cellular structures. In all cases the drug reduced mechanical strength of examined objects. For the leukemic cells the drug subcellular distribution was predominantly nuclear with some particulate cytoplasmic fluorescence. In erythrocytes, doxorubicin showed fluorescence mainly in cytoplasm and plasma membrane. The lowering of blast cells stiffness may be due to the interaction of doxorubicin with nuclear structures, especially with nucleic acids, as our studies with DNA confirmed. In addition, it is known that DOX inhibits the polymerization of actin and thus cytoskeletal modification may also be important in reducing of cell mechanical strength. In the case of erythrocytes - the non-nucleated cells, a significant effect on the decrease of cell stiffness, besides the cytoskeleton, may have the interaction of the drug with the cell membrane. Experiments with model phospholipid membranes confirmed that observed increase in cell elasticity originates, among other things, from the drug incorporation in the lipid membrane itself. The lowering of mechanical strength of leukemic cells may have an significant impact on the effectiveness of chemotherapy. However, the fact that doxorubicin interacts not only with proliferating cancer cells, but also with the health ones may explains the high toxicity of the drug at the therapeutic concentrations. Our observations also suggest that chemotherapy with doxorubicin may decrease the risk of vascular complications in acute leukemia, due to increasing the cell elasticity.

Evaluation of a linear birefringence measurement method with increased sensitivity.

The evaluation of a measurement method of linear birefringence with increased sensitivity is presented. The examined method is based on a substantial change of the geometrical phase caused by a small change of an examined medium's birefringence. The measuring setup consists of a linear polarizer, Wollaston compensator, and circular analyzer. The measurement is performed by tracking the phase shift of a fringe pattern. Specific orientation of the elements modifies the setup's response-the sensitivity of the setup can be controlled in a limited measuring range. The present paper concentrates on the factors affecting the setup's sensitivity as well as the accuracy of obtained results. The validity of the proposed approach has been demonstrated by measuring a phase retardance introduced by the liquid crystal retarder. A thousandfold increase in sensitivity has been obtained in the presented experiments, which allows the measurement of retardance introduced by the linear birefringent medium with an accuracy of 0.003° within the limited measuring range.

Time-series methods in analysis of the optical tweezers recordings.

In this paper we treat optical tweezers as discrete-time linear filters and analyze the recorded trajectories of the trapped beads using time-series methods. Using these techniques we obtain a simple analytical formula for the aliased power-spectrum density. Moreover, we separate influences of the noise and blur induced by the video camera from the physical content of the measurements, providing simple tools to detect and account for these distortions. Finally, checking how our tools work on the real data, we identify what parameters of video camera calibration the blur is dominating and what the additive noise is dominating. We also detect a range where these two distortions cancel each other so that the data can be mistakenly classified as undisturbed.

Moving average process underlying the holographic-optical-tweezers experiments.

We study the statistical properties of recordings that contain time-dependent positions of a bead trapped in optical tweezers. Analysis of such a time series indicates that the commonly accepted model, i.e., the autoregressive process of first-order, is not sufficient to fit the data. We show the presence of a first-order moving average part in the dynamical model of the system. We explain the origin of this part as an influence of the high-frequency CCD camera on the measurements. We show that this influence evidently depends on the applied exposure time. The proposed autoregressive moving average model appears to reflect perfectly all statistical features of the high-frequency recording data.

Cluster formation in ferrofluids induced by holographic optical tweezers.

Holographic optical tweezers were used to show the interaction between a strongly focused laser beam and magnetic nanoparticles in ferrofluid. When the light intensity was high enough, magnetic nanoparticles were removed from the beam center and formed a dark ring. The same behavior was observed when focusing vortex or Bessel beams. The interactions between two or more separated rings of magnetic nanoparticles created by independent optical traps were also observed.

New scanning technique for the optical vortex microscope.

In the optical vortex microscopy the focused Gaussian beam with optical vortex scans a sample. An optical vortex can be introduced into a laser beam with the use of a special optical element--a vortex lens. When moving the vortex lens, the optical vortex changes its position inside the spot formed by a focused laser beam. This effect can be used as a new precise scanning technique. In this paper, we study the optical vortex behavior at the sample plane. We also estimate if the new scanning technique results in observable effects that could be used for a phase object detection.

Adjustment method of an imaging Stokes polarimeter based on liquid crystal variable retarders.

The description of adjustment of an imaging Stokes polarimeter constructed and tested in our laboratory is presented. Our polarimeter's operation is based on six fast intensity distribution measurements realized in six different configurations of linear and circular analyzers. Using liquid crystal variable retarders (LCVRs) makes this construction compact and mechanically simple. However, new problems arise with proper azimuthal alignment as well as with proper LCVR voltage adjustment. Three basic steps of the adjustment procedure adapted to the specific construction of our polarimeter are described in detail. Some remarks concerning the critical parameters of the used CCD camera's parameters are also presented, as well as experimental verifications of the setup's accuracy acquired due to the proper adjustment process.

Micro-step localization using double charge optical vortex interferometer.

We investigate the diffraction effects of focused Gaussian beams yielding a double optical vortex by a nano-step structure fabricated in a transparent media. When approaching such a step the double vortex splits into single ones which move in a characteristic way. By observing this movement we can determine the position of the step with high resolution. Our theoretical predictions were verified experimentally.

Dynamic polarization states and birefringence distributions measurements in spatial elliptical polariscope using Fourier analysis method.

A new method to measure the light polarization state and the birefringent media parameters is proposed. We have used the setup described previously, consisting of two pairs of the linear Wollaston and circular compensators which form a set of two spatially modulated elliptical compensators. We have modified this setup introducing some carrier frequencies in all compensators and assuming that the second linear one would introduce the frequency which is a multiplicity of the basis frequency of the first linear compensator. Both of these modifications allow calculating all polarization parameters of polarized light or birefringent medium from only one measured intensity distribution of the light outcoming the described setup. They allow measuring not only the parameters of homogeneous beams/mediums but also x,y-distributions of all desired parameters, like azimuth and ellipticity angles of the light or first medium eigenvector and the phase difference introduced by this medium. The proposed calculation method comprises of Fourier analysis of obtained intensity distribution with some manipulation of coordinate system and filtration of obtained data. This method is claimed to be simple and fast enough to be treated as a real-time method.

Transmission imaging polarimetry for a linear birefringent medium using a carrier fringe method.

We present an imaging polarimeter in transmission mode that is based on a carrier frequency method and allows a spatially resolved polarimetric description of nondichroic linear birefringent media. The apparatus incorporates a generator of polarization states in the incoming pathway and a Wollaston prism and a linear polarizer as the analyzer unit. A series of two fringe pattern images of the birefringent sample under study, corresponding to two independent polarization states of the generator unit, were recorded. From these images and by using Fourier analysis, the 2D distribution of azimuth angle and retardation were calculated. Two alternative generator units were used: (i) a linear polarizer combined with a rotatory quarter-wave plate and (ii) a liquid-crystal variable retarder. A uniform quarter-wave plate at different orientations was measured with both generator units to demonstrate the effectiveness and the accuracy of the method. The mean absolute deviations were 1.8 degrees and 4.1 degrees for the azimuth and the retardation, respectively, with the first generator unit, and 2.9 degrees and 4.4 degrees for the second one. Moreover, some nonuniform birefringent samples presenting wider ranges of azimuth and retardation were also tested.

Application of space periodic variation of light polarization in imaging polarimetry.

The application of space periodic variation of light polarization for measurement and calculation of the distribution of the phase retardation between two eigenwaves propagating inside a linearly birefringent media and the distribution of the azimuth angle of the first eigenvector is described. The measuring method proposed does not require any mechanical movements or rotations of any optical elements. Application of a liquid crystal (LC) modulator instead of a quarter-wave plate gives an opportunity to introduce the required phase shift. The space periodic modulation of the polarization of light is achieved by the use of a Wollaston prism placed inside the path of the light beam. Then a fast Fourier transform is used for further calculations. The number of measurements of the light intensity at the output of the system is minimized to two. These assumptions make the proposed method very fast, which is especially important in measurements of the objects with optical anisotropy that is changing in time.